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Position: Home > Articles > Development of Polymorphic SSR Markers in Tobacco Based on RAD Sequencing Chinese Tobacco Science 2018 (1) 1-9

基于RAD-seq数据开发烟草多态性SSR标记

作  者：
李海洋;李荣华;夏岩石;袁清华;张振臣;赵伟才;郭培国
单  位：
广东省农业科学院作物研究所;广东省烟草南雄科学研究所;广州大学生命科学学院
关键词：
烟草;RAD-seq;SSR标记;多态性
摘  要：
采用RAD-seq技术对两份烟草材料"118-3"和"粤烟98"进行简化基因组测序,分别获得45 119 346和50 360 738个高质量干净读长(HQ clean reads)。通过序列分析,在22 145个reads覆盖的参考基因组序列中共检测到25 343个SSR位点,其中以二和三核苷酸重复基序最为丰富,占总SSR位点的71.59%;除单核苷酸类型外,SSR基序的重复次数主要集中在4~9之间。根据SSR位点两翼的序列,利用Primer 3.0成功设计出23 346对SSR引物,引物设计的成功率为92.12%。依据测序数据初步发现这些SSR中有323个(1.38%)SSR在两份测序材料间具有多态性,随机选择其中50个SSR在4份烟草材料中进行验证,结果发现在两份测序材料间的多态率为76%,存在24%的假阳性;在另两份烟草材料中多态率为20%。研究结果表明,利用简化基因组测序技术能够开发大量SSR标记和发现样品间具有多态性的SSR标记,可为SSR标记的开发和应用提供基础。
译  名：
Development of Polymorphic SSR Markers in Tobacco Based on RAD Sequencing
作  者：
LI Haiyang;LI Ronghua;XIA Yanshi;YUAN Qinghua;ZHANG Zhenchen;ZHAO Weicai;GUO Peiguo;School of Life Sciences, Guangzhou University;Crop Research Institute, Guangdong Academy of Agriculture;Nanxiong Research Institute of Guangdong Tobacco Co., Ltd.;
关键词：
tobacco;;RAD-seq;;SSR marker;;polymorphism
摘  要：
Two tobacco genotypes, "118-3" and "Yueyan 98", were sequenced using restriction-site associated DNA(RAD) on the Illumina Mi Seq PE300 platform, and 45 119 346 and 50 360 738 HQ clean reads were obtained in "118-3" and "Yueyan 98", respectively. Through sequences analysis, 25 343 SSR loci were identified in the reference genome sequences covered by 22 145 reads. Of these SSR loci, di-and tri-nucleotide repeat motifs were the two most abundant types, with the percentage of 71.59% in total SSR loci. Apart from mono-nucleotide type, the repeat counts of SSR motifs mainly ranged from 4 to 9. 23 346 primer pairs(92.12% of total SSR loci) were successfully designed using the Primer 3.0 software based on the bilateral sequences of SSR loci, and 323(1.38%) of them showed polymorphism between the two genotypes according to the sequencing data. Fifty of the 323 primer combinations were randomly selected to verify their polymorphisms in 4 tobacco genotypes, the results showed that 76% of the primer pairs were polymorphic between the two sequenced genotypes, and the false positive rate was 24%; and the polymorphic rate in the other two genotypes was 20%. These results indicate that RAD sequencing could be used to develop a large number of SSR markers especially polymorphic SSR markers, which could provide basis for the development and application of SSR markers.

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